Trypanosoma cruzi is a flagellate protozoal parasite, a member of the order Kinetoplastida and of the family Trypanosomatidae, which is responsible for Chagas disease which affects naturally millions of persons, mainly in Latin America.
In vertebrate hosts, Trypanosoma cruzi is present in two forms: one which is mobile by means of its flagellum or trypomastigote and which does not divide; the other is aflagellate, or intracellular amastigote, which multiplies by binary division.
Transmission of the protozoan in man occurs through hematophagous insects of the family Reduviidae, during a blood meal followed by dejections at the site of the bite. The vector insect thus releases the infectious metacyclic trypomastigote forms which will colonize many cell types through the blood circulation. Trypanosoma cruzi infects cardiac and skeletal muscular cells, the glial cells and the cells of the mononuclear phagocytic system. After passive penetration into the host cell, the trypomastigote form of the parasite differentiates into the amastigote form, divides actively and then this is followed by a release of the trypomastigote forms, thereby causing a new cell invasion.
The insects will complete the parasitic cycle by ingesting, during a blood meal, the trypomastigote forms in the host. The latter differentiate into epimastigote forms in the vector's middle intestine and finally into the infectious metacyclic trypomastigote forms in the posterior intestine.
Two phases can be distinguished in the Chagas disease: the acute phase and the chronic phase. The acute phase occurs after a transfusional, congenital or vectorial type contamination and lasts for a few weeks. It is characterized by a large number of parasites circulating in the blood and corresponds to an exponential division of the protozoan. The acute phase is most often asymptomatic. However, in infants contaminated by their mother, the acute phase, which is marked by an acute cardiopathy, may be critical. The chronic phase may extend over many years. In some individuals, this phase is asymptomatic. On the other hand, other patients have tissue lesions in the heart or digestive type manifestations. In any case, clinical diagnosis must always be confirmed by tests for the detection either of antibodies directed against the parasitic antigens, or of the parasite itself.
This disease is becoming a worldwide problem because of the contamination through blood transfusion. It was therefore becoming essential to have available diagnostic tests which make it possible to determine the presence of the parasite in individuals. Various serological tests include direct agglutination, indirect immunofluorescence (IIF), complement fixation tests (CFR), ELISA tests (Enzyme Linked Immunosorbent Assay). The Trypanosoma cruzi antigens used for the serological tests are obtained from a total lysate of the noninfectious stage of the parasite or from partially purified protein fractions. However, these fractions do not allow antigens to be obtained in sufficient quantity and quality for the production of a reliable serological diagnostic test. Furthermore, the complexity of the parasite and the strain-to-strain antigenic polymorphism introduce an additional difficulty in the reproducibility of the different preparations. Finally, there are many risks of cross-reactivity with other protozoa, more particularly with Trypanosoma rangeli, a nonpathogenic parasite, and the family Leishmania. Another disadvantage of these techniques is the absence of determination of the disease phase which would allow a treatment from the onset of the acute phase.
In order to solve these various problems, it was envisaged to produce a serological diagnostic kit composed of recombinant proteins which would be specific for Trypanosoma cruzi.
Various research groups have screened libraries for expression of Trypanosoma cruzi genomic DNA or complementary DNA in the vector .lambda.gt11, using sera from patients suffering from Chagas disease. The .lambda.gt11 phage allows the insertion of foreign DNA of a maximum size of 7 Kb into the EcoR1 site localized in the lacZ gene, under the control of the lac promoter. The product obtained is a recombinant protein used with beta-galactosidase, which is inducible by IPTG (isopropyl beta-D-thiogalactoside).
Various Trypanosoma cruzi genes, encoding proteins recognized by the Chagasic sera were thus characterized. Among the recombinant antigens described, the H49 antigen may be mentioned (Paranhos et al., 1994 (1)). However, this antigen does not allow a serological detection sensitivity of 100% of the patients in the acute or chronic phase. It was therefore envisaged to combine the H49 antigen with the CRA antigen (Cytoplasmic Repetitive Antigen) (Lafaille et al., (1989) (2)) but still without solving this problem.